Toxicity of aromatic compounds, antibiotics, organic solvents and bacteriocidic agents to microorganisms presents a major problem in the field of microbiology. In addition, tolerance to pHBA, antibiotics, aromatic compounds, parabenes and aromatic amino acids are of significant importance in various biotechnology areas such as biotransformation, biodegradation, food, pharmaceuticals, and cosmetics. Factors influencing tolerance appear to be varied and not always understood. Increasingly, attention has turned to genetic manipulation to create microbes that are able to thrive in high concentrations of aromatic compounds and organic solvents or to create microbes that are more sensitive to antibiotics and bacteriocidic agents, e.g., parabene preservatives. Among these are microbes that can synthesize monomers that can be used for the ulterior synthesis of added value polymers. One of these products of interest is pHBA that can be synthesized from toluene as described below. para-Hydroxybenzoic acid (pHBA) is a key monomer for production of liquid crystal polymers, (e.g., Zenite® that are used in board displays of computers and other electronic devices and for parabene preservatives).
A current limitation on the biotransformation of toluene into pHBA is the relative toxicity of these compounds for cells (Sikema et al., Microbiol. Rev. 50:201-222 (1995), as well as the toxicity of the product being produced (WO 9856920). One enzymatic pathway of increasing commercial interest for biotransformation is that of toluene degradation through the toluene monooxygenase pathway (TMO pathway). This pathway includes the following steps: toluene is oxidized to p-cresol with toluene monooxygenase, p-cresol is progressively oxidized to p-hydroxybenzyl alcohol and p-hydroxybenzaldehyde with p-cresol methylhydroxylase, and p-hydroxybenzaldehyde is then oxidized to p-hydroxybenzoic acid (PHBA) with p-hydroxybenzaldehyde dehydrogenase and pHBA is further oxidized to protocatechuic acid (PCA) with p-hydroxybenzoate hydroxylase. PCA is further metabolized to the TCA cycle where it is used for cell biosynthesis or energy metabolism.
Bacteria that possess the TMO pathway are useful for the degradation of toluene and other organics and are able to use these as their sole source of carbon (Wright et al., Appl. Environ. Microbiol. 60:235-242 (1994); Duetz et al., Appl. Environ. Microbiol. 60:2858-2863 (1994); Leahy et al., Appl. Environ. Microbiol. 62:825-833 (1996)). Bacteria that possess the TMO pathway are common among the genus Pseudomonas and species known to possess it include Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Pseudomonas mendocina. 
Recently, various strains of Pseudomonas possessing the TMO pathway have been used to produce muconic acid from toluene via manipulation of growth conditions (U.S. Pat. No. 4,657,863; U.S. Pat. No. 4,968,612). Additionally, strains of Enterobacter with the ability to convert p-cresol to p-hydroxybenzoic acid (PHBA) have been isolated from soil (JP 05328981). Further, JP 05336980 and JP 05336979 disclose isolated strains of Pseudomonas putida with the ability to produce pHBA from p-cresol.
Amaratunga et al., (U.S. Pat. No. 6,030,819) reported a method for fermentation of glucose to PHBA through the shikimic acid pathway using genetically engineered E. coli. These workers provided a plasmid which controls the overexpression of chorismate pyruvate lyase, the bacterial enzyme which catalyzes the production of pHBA from chorismate. The yield reported, concentration and time were 0.04 g pHBA per g glucose, 6.2 g/L and 40 hr, respectively. These values do not make this process very attractive.
Inactivation of pobA gene(s) that codes for p-hydroxybenzoate hydroxylase allow production of pHBA from toluene or p-cresol in microorganisms that posses the TMO pathway with complete conversion of these substrates to pHBA at high conversion yield and titer (WO 9856920). Inhibition of cell growth, pHBA production and the activity of the enzymes in the metabolic toluene degradation pathway in the presence of high concentrations of pHBA has been known for many years (Eklund T., Int. J. of Food Microbiol. 2, 159-167 (1985)). A fermentation method for the biological production of pHBA has-been developed (WO 9856920). However, the successful production of pHBA to high concentrations requires increasing the tolerance to pHBA.
Overexpression of an efflux system or its expression from a plasmid vector results in increased resistance of bacteria to a variety of toxic substances, while inactivation of an efflux system causes an increase in sensitivity to antibiotics and toxic substances (Li et al., J. Bacteriol. 180:2987-2991 (1998); Ramos et al., J. Bacteriol. 180:3323-3329 (1998)). Such efflux systems are increasingly being recognized in a wide range of bacteria, particularly gram-negative ones.
TonB-dependent energy transduction can be considered an all-purpose system for the delivery of energy to the outer membrane. This system is widely distributed among gram-negative bacteria (see list below), where it also energizes the transport of iron-siderophores and vitamin B12 (Jarosik et al., Infect. Immun. 62:2470-2477 (1994); Torres et al., Mol. Microbiol. 23:825-833 (1997)). The TonB-dependent energy transduction complex consists of, at least, three proteins, TonB, ExbB, and ExbD. Although ExbB and ExbD are essential for TonB activity, TonB functions as the true energy transducer that couples the proton motive force of the cytoplasmic membrane to drive active transport at the outer membrane (Karlsson et al., Mol. Microbiol. 8:379-388 (1993); Braun, FEMS Microbiol. Rev. 16:295-307 (1995); Letain et al., Mol. Microbiol. 24:271-283 (1997); Kadner, Mol. Microbiol. 4:2027-2033 (1990); Postle, J. Bioenerget. Biomemb. 25:591-601 (1993); Moeck et al., Mol. Microbiol. 28:675-681 (1998)).
Microorganisms Containing exbB, exbD and tonB GenesAquifex aeolicusBordetella bronchisepticaCampylobacter coliChlamydia pneumoniaeChlamydia trachomatisEnterobacter aerogenesEscherichia coli K-12Escherichia coli MG 1655Haemophilus ducreyiHaemophilus influenzaeHelicobacter pylori 26695Helicobacter pylori J99Kiebsiella pneumoniaeNeisseria gonorrhoeaeNeisseria meningitidisPasteurella haemolyticaPseudomonas aeruginosaPseudomonas putida DOT-T1EPseudomonas putida WCS358Salmonella typhimuriumSerratia marcenscensVibrio choleraeXanthomonas campestrisYersinia enterolitica
While tonB genes and organization of the exbB and exbD, and tonB genes have been identified and characterized in a number of microorganisms, no prior art has been found which describes that tonB genes are involved in tolerance to a wide variety of chemicals such as pHBA, antibiotics, aromatic compounds, parabenes, and aromatic amino acids. Moreover, no prior work has disclosed that tonB mutants are sensitive to the above chemicals. It has previously been disclosed that tolerance to aromatic hydrocarbons involves the operation of the efflux pumps that remove toxic compounds from the membranes (Ramos et al., J. Bacteriol. 180:3323-3329 (1998) and Mosqueda et al., Gene 232:69-76 (1999)).
In summary, the current methods available to produce PHBA, aromatic compounds, antibiotics, aromatic amino acids, and bactericidal agents suffer from serious disadvantages which make their commercial production too expensive. Therefore, there remains a need to understand the mechanisms of tolerance in order to design better antibiotics, bacteriocidic and bacteriostatic agents, and to generate microbes with enhanced biocatalytic potential which can tolerate large amounts of the above mentioned compounds at a commercially reasonable cost. Moreover, there is a crucial need for controlling microbial tolerance to pHBA, aromatic compounds, antibiotics, aromatic amino acids, and bactericidal agents to allow their effective production, effective removal and further help to increase or decrease the effect of certain chemicals in a microbial cell growth.